EKSTRAKSI GLUKOMANAN DARI TEPUNG PORANG

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Ekstraksi Glukomanan dari Tepung Iles-Iles Kuning … (Nurlela, dkk.)

EKSTRAKSI GLUKOMANAN DARI TEPUNG PORANG

(Amorphophallus muelleri Blume) dengan Etanol

Extraction of Glucomannan from porang (Amorphophallus muelleri Blume) flour using Ethanol

Nurlela*, Dewi Andriani, Ridha Arizal

Program Studi Kimia, Fakultas MIPA, Universitas Nusa Bangsa

Jl. K.H. Sholeh Iskandar Km.04 Cimanggu, Tanah Sareal, Bogor, 16166 *e-mail: [email protected]

ABSTRAK

Iles-Iles kuning (Amorphophallus muelleri Blume) adalah sumber potensial glukomanan, suatu senyawa polisakarida yang memiliki beberapa sifat khusus yang sering digunakan di berbagai bidang industri, farmasi, dan makanan. Kualitas glukomanan yang diproduksi di dalam negeri masih belum dapat menyamai kualitas glukomanan impor. Penelitian ini bertujuan untuk mengetahui pengaruh perbedaan metode ekstraksi glukomanan dari tepung iles-iles kuning menggunakan etanol untuk mendapatkan kadar glukomanan yang tinggi dengan kualitas yang lebih baik. Glukomanan yang baik memiliki viskositas tinggi dan kandungan air, abu, protein, lemak, dan pati yang rendah. Ekstraksi tepung iles-iles kuning menggunakan etanol konsentrasi bertingkat (40, 60, dan 80%) mampu menghasilkan kadar glukomanan lebih tinggi dan kualitas lebih baik daripada etanol 60% dengan tiga kali ulangan. Ekstraksi menggunakan etanol bertingkat mampu meningkatkan kadar glukomanan dari 16,43% menjadi 62,2%. Spektra Fourier Transform Infra Red (FTIR) dari glukomanan hasil ekstraksi menunjukkan adanya gugus-gugus penyusun senyawa glukomanan (O-H, C=O, C-O, C-H) seperti yang ditunjukkan juga pada spektra glukomanan komersil.

Kata Kunci: Amophophallus muelleri Blume, tepung iles-iles, glukomanan, ekstraksi konsentrasi bertingkat, etanol.

ABSTRACT Iles-Iles kuning (Amorphophallus muelleri Blume) is a potential source of glucomannan, a polysaccharide compound that has several special properties which often used in various fields of industry, pharmacy, and food. The quality of glucomannan produced domestically still cannot match the quality of imported glucomannan. This study aims to determine the effect of difference extraction methods of glucomannan from iles-iles kuning flour using ethanol to obtain high contain of glucomannan with better quality. Good quality of glucomannan has high viscosity and contains small amount of water, ash, protein, fat, and starch. Extraction of iles-iles kuning flour using multilevel concentration of ethanol (40, 60, and 80%) was able to produce higher glucomannan and better quality than ethanol 60% with three times of extraction. Extraction using multilevel ethanol was able to improve glucomannan content from 16,43% to 62,2%. Fourier Transform Infra Red (FTIR) spectra of extracted glucomannan showed the functional groups composing the glucomannan compound (O-H, C=O, C-O, C-H) similar to the spectra of commercial glucomannan.

Keywords: Amophophallus muelleri Blume, iles-iles flour, glucomannan, multilevel concentration extraction, ethanol.

mailto:[email protected]

Sains dan Terapan Kimia, Vol. 14, No. 2 (Juli 2020), 88 – 98

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INTRODUCTION

The tuber of iles-iles kuning

(Amorphophallus muelleri Blume) is a very

potential source of glucomannan because it

contains high glucomannan (Yanuriati et al.,

2017). Glucomannan is a polysaccharide

consisting of units of D-glucose and D-

mannose (Saputro et al., 2014). Glucomannan

has been used since ancient times as food

and medicine in China and Japan, and even

now its use is increasingly widespread in the

pharmaceutical, cosmetic and some chemical

fields. Koswara (2013) said that iles-iles

contain 5-60% of glucomannan, and iles-iles

in Indonesia generally have glucomannan

content of around 14-35%.

Iles-Iles kuning is a potential source of

glucomannan but the quality of glucomannan

produced domestically still cannot match the

quality of imported glucomannan. This can be

caused by the quality of glucomannan

produced in the country is still far from the

standard that should be used in various fields.

According to Mulyono (2010), increasing the

quality of glucomannan flour can be done by

multilevel extraction.

Table 1. Composition of Iles-Iles Kuning Flour Based on Literature

Component Iles-Iles Kuning Flour before Treatment

Commercial Glucomannan Flour

Moisture (%) 8,71 8,25 Ash (%) 4,47 0,37

Starch (%) 3,90 0,27 Protein (%) 2,34 0,63

Fat (%) 3,25 0,79 Viscosity (c.Ps) 3313 11000

Glucomannan (%) 43,99 92,51

(Source : Widjanarko et al., 2014)

Experiment of glucomannan extraction

from iles-iles kuning flour has been widely

carried out in Indonesia. Faridah & Widjanarko

(2013) using multilevel concentration of

ethanol for glucomannan extraction, i.e. first

extraction with 40% ethanol, then the

precipitated sample was extracted again with

ethanol 60%, and finally extracted with 80%

ethanol. The extraction with multilevel

concentration of ethanol resulted 79.26% of

glucomannan. Furthermore, Saputro et al

(2014) compared extraction using ethanol

solvents with different concentrations, which

was 40, 50, and 60%. The highest content of

glucomannan was produced from ethanol

60%, which was 64.22%. According to

Yanuriati et al. (2017), glucomannan content

will be higher if the extraction is repeated.

Fithri (2017) has also researched extraction of

iles-iles kuning flour with water, then washed

with 1-propanol and resulted 64.49% of

glucomannan content. However, 1-propanol is

not recommended because the price is more

expensive than ethanol. In addition, ethanol is

safe solvent if the processed product are

applied for food and health.

In this study, glucomannan was extracted

from iles-iles kuning flour using ethanol with

two different methods. The first method is

extraction using ethanol with multilevel

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concentration that is extracted with ethanol

concentration of 40% (stage I), then proceed

with extraction using ethanol 60% (stage II),

and finally extracted with ethanol 80% (stage

III), respectively one-time repetition in each

stage. Next, the second method is extraction

using ethanol 60% repetitively for three times

(fixed concentration).

MATERIAL AND METHODS

Equipment and Material

The primary materials used in this study

were three years old iles-iles kuning tubers

from the species Amorphophallus muelleri

Blume. The tubers were obtained from the

Bogor Agricultural University experimental

garden, Bogor, Indonesia. Ethanol 96%,

aquadest, sodium metabisuphite, H2SO4,

NaOH, HCl, hexane, D-glucose standard, 3.5

Dinitrosalicylic acid, boric acid, formic acid-

NaOH buffer, lugol reagent, potassium

phosphate buffer, and other chemicals were

analytical grade from Merck, Smart Lab, etc.

The equipments used in this study were

spectrophotometer UV-Visible Shimadzu,

Fourier Transform Infra Red Perkin Elmer

Spectrum Two, viscometer Brookfield DV-III,

magnetic stirrer, centrifuge, oven, and glass

ware.

Methods

Preparation of iles-iles flour

The tubers were washed first, then peeled

and sliced into ± 2 mm. The chips were

washed using water and soaked in 2000 ppm

of sodium metabisulphite solution for 20

minutes. The chips were dried in the oven for

16 hours at 65oC, then grinded using a disk

mill. Flour was sieved by sieve sized 60 mesh

and stored in a dry container (Aryanti & Abidin,

2015).

Extraction of Glucomannan

The first method is extraction of

glucomannan with multilevel concentration of

ethanol. Iles-Iles kuning flour was put into

ethanol 40% with ratio 1 gram flour /15 mL of

solvent and stirred for 1 hour then filtered with

cotton cloth, then the sample was extracted

again with ethanol 60%, and 80% with the

same treatment. The precipitate was dried in

oven at 45oC for 12 hours. Sample was

grinded and sieved in 60 mesh-sized. The

second method is extraction of glucomannan

using ethanol 60%, the ratio was 1 gram

flour/15 mL solvent and extracted for 1 hour.

The sample was filtered with cotton cloth. The

extraction was conducted for three times

repetitively. The precipitate was dried in oven

at 45oC for 12 hours. Sample was grinded and

sieved in 60 mesh-sized.

Physicochemical properties: colour and viscosity

The colour of glucomannan is directly

observed visually. The viscosity determined

using viscometer, sample was taken about 0.2

g and dissolved in 10 mL hot water. About 90

mL water added to the solution and the

viscosity was measured.


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Glucomannan content

The determination of glucomannan

content was carried out using the colorimetric

method with 3.5-Dinitrosalicylic acid reagents

(Chua et al., 2012). Glucomannan extract was

made by dissolving 0.2 g sample in NaOH-

formic acid buffer solution (0.1 mol/L, 100 mL)

and stirred for 4 hours. This solution was

centrifuged at 4000 rpm for 30 minutes. The

supernatant was glucomannan extract.

Glucomannan hydrolysate was made by

mixing 5 mL glucomannan extract with 2.5 mL

H2SO4 3M shaken with vortex and heated for

90 minutes in water bath, then the solution

allowed to cool in room temparature and

added 2.5 mL NaOH 6 M. The solution was

added with deionized water up to 25 mL, and

the solution formed was glucomannan

hydrolysate. Glucomannan extract and

hydrolyzate were measured by colorimetric

method, while deionized water was used as a

blank.

The standard of D-glucose (1 mg/mL) was

diluted to a solution of 0.10, 0.2, 0.30, 0.40,

and 0.50 mg/mL with deionized water. About 2

mL of standard solution was added with 3.5

DNS 1% (1.5 mL), then heated for 5 minutes

in boiling water, and added deionized water to

25 mL after it was cooled. The treatment of

glucomannan extract and glucomannan

hydrolysate was the same as the D-glucose

standard. These solutions are readily

absorbed in wavelength of 550 nm.

Glucomannan content was calculated by :

% Glucomannan = 5000 × f (5T-To)

m×

100

100-w

where f = correction factor, T = glucose

content of glucomannan hydrolysate (mg), To

= glucose content of glucomannan extract

(mg), m = mass of glucomannan (200 mg) and

w = water content of glucomannan

Chemical composition

Moisture content was determined by

weight difference after drying of samples,

following the official method of AOAC (2005).

Ash was determined gravimetrically (AOAC,

2005). Protein content was calculated from the

nitrogen content (N%: 6.25) analyzed by

Kjeldahl method (AOAC, 2005). Fat content

was determined using a Soxhlet apparatus

according to SNI 06-6989.10-2004. Crude

fiber was determined using acid-base

extraction according to SNI 01-2891-1992.

The starch was analyzed qualitatively by

staining glucomannan granules using I2-KI.

Characterization using Fourier Transform Infra Red (FTIR)

Specific functional groups of

glucomannan was determined using FTIR-

UATR. Spectrum sample was read in the

range 4000-400 cm-1.

RESULT AND DISCUSSION

Preparation of Iles-Iles Kuning Flour

Iles-iles kuning tubers soaked using

sodium metabisulfite 2000 ppm to prevent

browning process. Browning is caused by the

presence of carotene content, the enzyme

polyphenol oxidase (PPO) and polyphenolic

compounds including tannins (Zhao et al.,

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2010). Starch, calcium oxalate, and

temperature also affect the brightness of flour

(Sumarwoto, 2007). To prevent browning, in

this study iles-iles kuning chips soaked first

using sodium metabisulfite.

Figure 1. Iles-Iles Kuning Chips after Soaked in Sodium Metabisulfite.

Chips that have been soaked with sodium

metabisulfite did not browning (Figure 1).

Sodium metabisulfite is a reducing compound,

sulfite ions in this compound work to inhibit

non-enzymatic browning because the

carbonyl group will react with sulfite (Lubis et

al., 2004). According to Wedzicha et al (1984),

sulphur (IV) oxospecies could inhibit browning

or Maillard reactions during storage on

vegetables. In Dwiyono's research (2014),

soaking iles-iles kuning tubers in sodium

metabisulfite solution increase the brightness

without reducing glucomannan content.

Converting iles-iles kuning tubers into

flour is to reduce high water content.

According to Koswara (2013), the water

content of iles-iles tubers is relatively high,

between 70-80%. This situation causes during

storage of glucomannan will be damaged by

enzyme activity. In addition, the minimum

water content limit at which microorganisms

can still grow is around 14-15% (Fardiaz,

1989).

Colour

The color of glucomannan extract showed

in Figure 2. It can be seen that the yellow color

of the samples produced after extraction was

fainter than iles-iles kuning flour. Meanwhile,

the glucomannan exctract from method I was

faded more than method II. Iles-iles kuning

tubers have carotene content of around 40

mg/kg, and the compound caused iles-iles

kuning tubers have yellow color (Wootton et

al., 1993).

Figure 2. Difference Colour of Glucomannan Extract; (1) Extraction Using Multilevel Concentration of Ethanol; (2) Extraction Using Fixed Concentration of Ethanol; (3) Iles-iles Kuning Flour

1 2 3


93

Carotene is a carotenoids, which are

soluble in fat or organic solvents but insoluble

in water. Therefore, the higher concentration

of ethanol used, the more soluble the

carotene. Ethanol concentration in method I

which was higher than ethanol concentration

in method II causes the carotene contained in

flour to dissolve more so that the color of

glucomannan extraxted by method I becomes

brighter.

Composition of glucomanan

Extraction of iles-iles kuning flour was

carried out using ethanol with two different

methods, the first method was extraction using

multilevel concentration of ethanol (40, 60,

80%) and the second method was using fixed

concentration of ethanol 60% for three times

repetitively. The comparison of

physicochemical composition of glucomannan

showed in Table 2.

Table 2. Composition of Glucomannan Extract

Parameter Iles-Iles Kuning Flour

before treatment Sample of

Extraction Method I

Sampel of Extraction Method II

Glucomannan (%) 16,43 62,2 43,02 Viscosity (cP.s) - 4024,14 549,88 Moisture (%) 8,32 10,30 10,63 Ash (%) 3,37 0,52 1,39 Protein (%) 7,13 4,86 5,96 Fat (%) 0,3 <0,0001 <0,0001 Crude Fiber (%) 3,54 3,97 3,69 Starch* +++ + ++

+ = blue colour

Iles-iles kuning flour extraction using

multilevel concentration of ethanol resulted

higher glucomannan content than extraction

using ethanol with fixed concentration. This is

caused by more impurities dissolved in

extraction method I than methode II. These

impurities have different solubility in ethanol.

Polarity of solution is determined from its

dipole moment. Dipole moment of water is

1.84 Debye, greater than ethanol which is

about 1.69 Debye. The polarity of ethanol 40%

is higher than ethanol 60% and 80%, because

40% ethanol consists of 40% of ethanol and

60% of water. Ethanol 40% would dissolve

more polar compounds, such as protein and

sugar (Xu et al., 2014). Ethanol 60% dissolves

compounds that are insoluble in ethanol 40%,

like starch (Nurlela et al., 2019). Ethanol 80%

has a lower polarity so that it would dissolve

compounds with lower polarity as well as fat,

calcium oxalate, and ash (Kurniawati &

Widjanarko, 2010). This method can maximize

the extraction of glucomannan and

glucomannan with high purity can be

produced. According to Widjanarko and

Megawati (2015), the low value of

glucomannan yields can be caused by

imperfect coagulation or precipitation

processes. The process of coagulation are

affected by three factors, heating, stirring, and

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the addition of electrolytes. According to

Nindita et al. (2012), the extraction time that is

not optimal can cause the flour not to be

extracted properly.

In this study, viscosity of method I and II

were 4024.14 cP and 549.88 cP, respectively.

According to Faridah (2016), glucomannan

has a very high molecular weight (> 300 kDA)

which can produce a very thick liquid. The

viscosity of the extracted glucomannan was

closely related to the glucomannan content

contained in the flour. Higher glucomannan

content would increase the viscosity of the

flour. Glucomannan is able to absorb water

and expand until it reaches 138-200% of the

initial weight of flour and occurs quickly. Its

ability to absorb water affected the viscosity

(Kurniawati & Widjanarko, 2010). The value of

viscosity from this study is still low compared

to the value of viscosity from the study of

Widjanarko et al (2011), which is about 9833

cP.s. It can be caused by the change in

amorphous form of glucomannan during

extraction process using ethanol. The

amorphous form changes from easily

dissolved in water to crystalline form that is

difficult to dissolve in water, so the viscosity

value decreases (Kato & Matzuda, 1969). The

low viscosity value can also be caused by the

large particle size, making it difficult to smooth

it with a blender or pestle because of its very

hard texture (Mulyono, 2010).

Moisture content is one of the most

important characteristics in food because

water can affect the appearance, texture, and

taste of food ingredients. High moisture

content can damage the freshness of food

because it is easier for bacteria, mold and

yeast to grow (Yusuf et al., 2016). The

minimum moisture level limit where

microorganisms can still grow is around 14-

15% (Fardiaz, 1989). From the test results

obtained all glucomannan extracts have water

content below 14%, which ranges from 8-11%.

Compounds that attach to glucomannan

such as ash, fat, protein, and starch are

considered impurities because they can affect

the special characteristic of glucomannan.

Starch that is attached to glucomannan

decrease the strength of the gel formed of

glucomannan. The presence of starch in

glucomannan also results in decreasing the

viscosity of glucomannan solution (Fadilah,

2017). Table 2 showed the decrease in

impurity content after extraction using ethanol.

The impurities content in glucomannan extract

using multilevel concentration of ethanol

(method I) was lower than using ethanol with

fixed concentration (method II). The impurities

present in the extracted glucomannan flour

have different polarity, so that multilevel

concentration of ethanol would be more

effective in separating them. The presence of

dark blue color after staining indicated high

starch content of glucomannan. Lugol's

solution (KI-I2) would not detect simple sugars

such as glucose or fructose (Libretext, 2019).

The result showed yellowish blue indicating

that the glucomannan only contained little

starch.


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Characterization of Glucomannan

The extract glucomannan characterized

by FTIR to determine the functional groups of

glucomannan. Figure 3 showed the spectrum

of glucomannan samples.

Figure 3. FTIR Spectra of Extract Glucomannan; (1) Method I; (2) Method II; (3) Commercial Glucomannan

1

3

2


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Glucomannan extracted from method I

and method II showed O-H at peak of 3315

cm-1 and 3320 cm-1 respectively. The peak at

3000-3700 cm-1 showed the O-H group on the

glucomannan structure. This was consistent

with the statement of Zhang et al. (2001)

stating that the glucomannan spectrum is

dominated by spectral bands related to the

stretching vibrations of O-H and water groups

in the range of 3396 cm-1. The peak at wave

number 2926 cm-1 and 2923 cm-1 were showed

the C-H group. The carbonyl group on acetyl

was clearly shown in wave number about 1739

cm-1 and 1736 cm-1 in the glucomannan flour

extracted by method I and method II.

Sastrohamidjojo (1992) stated that the

absorption of carbonyl group bonds (C=O)

was at wavenumber 1850-1630 cm-1. The C-O

group from ether can be seen at wavenumber

1230 cm-1 and 1247 cm-1, where the group will

give results at wave number 1260-1200 cm-1.

The absorption band at wavenumber 1019-

1016 cm-1 indicated the presence of COC

functional groups and this was supported by

the research of Setiawati et al. (2017) that the

COC function groups were present in

wavenumbers of 1020 cm-1, as well as the

opinion of Sastrohamidjojo (1992) which

stated that COC bond uptake give absorption

in the range of wave numbers 1300-1000 cm-

1.

The glucomannan spectrum from the

extraction method I and method II were almost

similar to the commercial glucomannan

spectrum, although there was a slight shift.

This could be caused by a number of

impurities present in the extracted

glucomannan in this study. Functional group in

protein, starch, fat, etc. could disturbed

glucomannan spectrum readings. The peaks

intensity of FTIR spectra of method II was

more similar to commercial glucomannan.

However, the commercial glucomannan that

we used does not provide information on its

glucomannan levels. Thus, it could not be

assumed that glucomannan extracted by

method II was purer than method I. The result

of the proximate test (Table 2) showed the

impurities (ash, protein, starch) of

glucomannan extracted by method I less than

method II, as well as glucomannan content

and viscosity of glucomannan extracted by

method I was higher than method II that

means method I can produce glucomannan

with higher levels with better quality.

CONCLUSIONS

From this study, the highest

glucomannan content (62.20%) was obtained

from iles-iles kuning flour by extraction with

multilevel concentration of ethanol 40, 60,

80% (method I). While the extraction method

with fixed concentration of ethanol 60%

(method II), resulting in 43.02% of

glucomannan content. Visual observation of

flour color, viscosity, and the proximate test

showed that method I gave better results than

method II. Determination of functional groups

using FTIR from extracted glucomannan flour

produce similar absorptions bands to

commercial glucomannan.

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ACKNOWLEDGMENT

We would like to thank Prof. Dr. Edi

Santosa (Bogor Agricultural University,

Indonesia) for providing of the iles-iles kuning

tubers used in this study.

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